INFECTION AND IMMUNITY, Oct. 1980, p. 297-3020019-9567/80/10-0297/06$02.00/0

Vol. 30, No. 1

Isolation and Characterization of Minicell-Producing Mutantsof Shigella spp.

P. GEMSKI* AND D. E. GRIFFINDivision of Communicable Diseases and Immun logy and Division of Biochemistry, Walter Reed Army

Institute of Research, Washington, D.C. 20012

Minicells are small, anucleate cells resulting from aberrant cell divisions at thepolar ends of bacilli. We have isolated minicell-producing mutant strains ofShigella flexneri 2a (MC-I) and Shigella dysenteriae 1 (MC-V) after mutagenesiswith N-methyl-N'-nitro-N-nitrosoguanidine. Microscopically, broth cultures ofMC-I and MC-V were found to contain free minicells, normal cells, and filamen-tous cells with polar, attached minicells. Both strains retained their ability toprovoke keratoconjunctivitis in guinea pigs and to invade HeLa cells. Purifiedsuspensions of minicells containing less than one whole cell per 106 minicells wereobtained by a combination of differential sedimentation and density gradientcentrifugation (5 to 30% [wt/vol] linear sucrose gradients). Each MC-I minicellcontained about 0.005 times the amount of deoxyribonucleic acid of one normalS. flexneri. The MC-V minicell had about 0.003 times the amount of deoxyribo-nucleic acid of one whole S. dysenteriae cell. Purified MC-V minicells weretreated with polymyxin B to release Shiga toxin. Shiga toxin was readily detectedin MC-V minicells by means of a microtiter HeLa cell cytotoxicity assay. Ourfindings indicate that such a minicell-producing alteration in the cell divisioncycle of shigellae has not significantly affected their virulence.

A mutant strain of Escherichia coli K-12 hasbeen shown by Adler et al. (1) to have an aber-rant cell division cycle which causes the produc-tion of small anucleate cells termed minicells.Since that report, minicell-producing mutants ofSalmonella, Bacillus, and other genera havebeen isolated (5). Studies have revealed thatsuspensions of minicells with less than one con-taminating parental cell per 106 minicells can beprepared by a combination of differential andrate-zonal sucrose density gradient centrifuga-tion (1, 5). Purified minicells, although lackingchromosomal deoxyribonucleic acid (DNA),contain normal levels of protein and ribonucleicacid (RNA) (1, 5). Although they are incapableof cell division, minicells possess functional cellwall, ribosomal, and metabolic systems (5).The uniqueness of the minicell system has

been utilized as a model for studying the celldivision cycle and the function and replicationof plasmids (5). Its use as a model for studyingfactors related to pathogenesis of diseases hasnot been exploited. Thus, we have initiated stud-ies to prepare and characterize minicell-produc-ing mutants of various pathogens known tocause enteric diseases. In the present report, wedescribe the isolation and characterization ofminicell-producing strains of Shigella flexneri2a and Shigella dysenteriae 1 which retain theirproperty of virulence.

MATERIALS AND METHODSBacterial strains. The S. flexneri 2a strain from

which a minicell-producing mutant was isolated wasM42-43, a virulent strain previously shown to elicitclassical dysentery in humans and other primates (4).S. dysenteriae 1 strain 1617, isolated during the recentepidemic of Shiga dysentery in Central America, wasemployed as the parent for constructing a minicell-producing mutant of S. dysenteriae 1. This strainreadily produces a toxin which possesses cytotoxic andenterotoxic properties (Shiga toxin); it is able to invadethe intestinal mucosa efficiently (7).Media. Penassay broth (Difco Laboratories), brain

heart infusion broth, and Trypticase soy agar (BBLMicrobiology Systems) were used for routine cultiva-tion of organisms. For cytotoxin assays, the organismswere grown in modified syncase (7), which containsbasic salts, 1% certified Casamino Acids (Difco Labo-ratories), 2% glucose, 0.004% tryptophan, and 0.004%nicotinic acid.

Mutagenesis. Bacteria, grown overnight at 37°Cin 10 ml of Penassay broth, were centrifuged andresuspended in 0.5 ml of fresh Penassay broth. Then0.1 ml of N-methyl-N'-nitro-N-nitrosoguanidine solu-tion (4.0 mg/ml) was added to the cell suspension.After 20 min of incubation at 37°C, 4.5 ml of additionalPenassay broth was added to the cell suspension.Incubation at 37°C was allowed to continue for an-other 5 h, at which time the bacterial suspensions werediluted and plated on Trypticase soy agar.

Sereny test for epithelial cell penetration (15).Drops of agar-grown bacterial suspensions (0.10 mlcontaining approximately 108 cells) were deposited

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into the conjunctival sacs of guinea pigs. The animalswere examined over a period of 72 h for evidence ofkeratoconjunctivitis.HeLa cell invasion test. As previously described

(9), HeLa cells (American Type Culture Collectionstrain CCL-2) were grown in Eagle minimal essentialmedium supplemented with 10% fetal calf serum, 20gnmol of glutamine, 50 ,ug of streptomycin, and 50 ,ug ofpenicillin G per ml and maintained in an atmosphereof 5% CO2 and 95% air. Trypsinized cells were used toprepare monolayers on cover slips placed on the bot-tom of sterile petri dishes. About 24 h before and againimmediately before infection of such monolayers withbacteria, the tissue culture medium was removed andreplaced with antibiotic-free medium. The monolayerswere infected to yield a final concentration of about 5x 107 bacteria per ml. After a 5-h incubation period,the monolayers were washed three times with Hanksbalanced salt solution and supplemented with freshantibiotic-free medium. Cover slips were removed at7 h postinfection, rinsed in saline, fixed in methanol-acetic acid mixture (3:1), and stained with Giemsastain.

Purification of minicells. Purified suspensions ofminicells were prepared by procedures similar to thosepreviously described (1, 5). Bacteria were grown inbrain heart infusion broth at 37°C with aeration for 24to 36 h. After a preliminary low-speed centrifugation(2,000 x g for 10 min) to remove a large proportion ofthe whole cells, the minicell-rich supernatant fluid wascentrifuged for 20 min at 20,000 x g. The resultingpellets, which contained both minicells and wholecells, were resuspended to yield a cell concentration ofabout 50- to 100-fold greater than the original culture.Samples (about 2.0 ml) of this suspension were thenlayered on 50-ml preformed sucrose density gradients(5 to 30% [wt/vol]) and centrifuged for 20 min at 3,000rpm in a Beckman L3 ultracentrifuge equipped withan SW 25.2 swinging-bucket rotor. This procedureresulted in the separation of a large, dense band ofminicells which was distinct from a secondary diffuseband found to consist mostly of whole cells. Theminicells were withdrawn from the gradient with asyringe, suspended in saline with gelatin, and sedi-mented at 20,000 x g for 15 min. A second and, on

occasion, third sucrose density gradient separation wasemployed to obtain highly purified preparations ofminicells. The level of contaminating whole cells wasdetermined by plating minicell preparations on Tryp-ticase soy agar. The number of minicells in purifiedsuspensions was determined by counting in a Petroff-Hausser chamber. Purified minicell suspensions, pre-

pared as described, usually contained less than one

whole cell per 106 minicells.Polymyxin B treatment. The polymyxin B pro-

cedure of Cerny and Teuber (3) adapted for the releaseof Shiga toxin from cells (D. E. Griffin, P. Gemski, andB. P. Doctor, Abstr. Annu. Meet. Am. Soc. Microbiol.1978, B48, p. 21) was employed. About 10'0 washedminicells were suspended in 0.14 M NaCl with andwithout polymyxin B sulfate (2 mg/ml) and incubatedat 37°C. Samples were removed at 2 and 60 min,centrifuged at 16,000 x g for 15 min, filter sterilized,and assayed for cytotoxicity to HeLa cells.HeLa cell cytotoxicity assay. The cytotoxicity of

preparations was assayed in HeLa cell monolayers bythe method of Gentry and Dalrymple (8).

RESULTS

Isolation of minicell-producing mutants.Minicell-producing mutants were isolated aftermutagenesis of parental strains with N-methyl-N'-nitro-N-nitrosoguanidine. Cells survivingmutagenesis were plated on Trypticase soy agar,and after 18 h of incubation at 370C, the colonieswere examined for an aberrant colonial mor-phology. We reasoned that minicell-producingmutants would produce irregular, rough-lookingcolonies due to their ability to form filamentouscells (5). Saline suspensions of such aberrantclones were then screened for the presence ofminicells by examination with a light-phase mi-croscope. Isolates which were filamentous orwhich produced small spherical cells were clonedand further tested to establish their minicell-producing capacity. Minicell-producing mutantsof S. flexneri 2a M42-43, designated MC-I, andof S. dysenteriae 1 1617, designated MC-V, wererecovered.Broth culture of both mutants yielded similar

findings with respect to cell morphology. Whenexamined with a light microscope, such cultureswere heterogeneous in that they contained cellswhich were either smaller or larger than wildtype, some cells which were filamentous, andfree minicells. Figure 1 shows a low-magnifica-tion phase-interference micrograph of live cellsfrom an exponentially growing culture of M42-43 MC-I. In addition to free minicells, a cellundergoing asymmetrical division to yield a min-icell is also shown. Examination of S. dysenter-iae 1 1617 MC-V gave similar findings.The purification of free minicells by means of

sucrose density gradient centrifugation is shownin Fig. 2. Such purified minicell suspensions ofMC-I and MC-V, when plated on Trypticase soyagar to determine the level of contaminatingwhole cells, were usually found to contain lessthan one whole cell per 106 minicells. The num-ber of minicells in such suspensions was deter-mined by the use of a phase-contrast microscopeequipped with a Petroff-Hausser counting cham-ber. The DNA content of purified MC-I andMC-V minicells (Table 1) was found to be smallas compared with that of whole cells.Virulence properties ofS. flexneri 2a MC-

I and S. dysenteriae 1 MC-V. Both minicell-producing mutants retained properties relatedto virulence of shigellae. Although their colonialmorphologies were typical of rough strains andthey tended to sediment when grown in liquidmedium, both strains agglutinated specifically inrespective antisera and were insensitive to rough

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MINICELLS OF SHIGELLA SPP. 299

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.4

FIG. 1. Phase-interference micrograph of live cells from an exponentialMC-I.

strain-specific phages (16) which only lysestrains defective in smooth lipopolysaccharidestructure. Both strains evoked a positive Serenytest for conjunctivitis (15), which reflects thecapacity of shigellae to penetrate epithelial cells.Their invasive ability was further illustrated

by the penetration of HeLa cells. About 5% ofHeLa cells were invaded within 7 h after bacte-rial infection was initiated, which was typical ofthe parental strains. Figure 3 shows such inva-sion of S. flexneri MC-I. Although the parentstrain M42-43 does not form filaments, the fila-ment-forming ability of MC-I, typical of manyminicell-producing mutants (5), is readily evi-dent. Free MC-I minicells were often seen in thecytoplasm of such infected HeLa cells. Althoughsimilar penetration of HeLa cells was detectedwith S. dysenteriae MC-V, degeneration of themonolayer was apparent at 7 h, a reflection ofthe cytotoxic activity of the Shiga toxin thatMC-V produces.

Purified minicells were also examined to de-termine whether they could invade HeLa cells.Even at infection doses of 1010 pure MC-I orMC-V minicells per 105 HeLa cells, no morpho-logical evidence of minicell penetration was seen.With purified MC-V minicells, however, cyto-toxic effects to the HeLa cell monolayer wereapparent, suggesting that individual minicellsretained significant levels of Shiga toxin. To

culture of S. flexneri M42-43

establish this, 1010 purified minicells of MC-Iand MC-V were treated with polymyxin B torelease Shiga toxin. The extracts were then as-sayed for toxin activity by using the HeLa cellcytotoxicity assay. As shown in Table 2, within2 min of polymyxin B treatment, significantrelease of toxin from pure MC-V minicells couldbe detected. No such toxin release was detectedwith MC-I minicells. This is not surprising sinceit has been shown that S. flexneri extracts con-tain at least 103-fold less toxin than similar prep-arations of S. dysenteriae 1 (13).

DISCUSSION

Minicell-producing mutants of S. flexneri 2aand S. dysenteriae 1 were recovered after mu-tagenesis with N-methyl-N'-nitro-N-nitrosogua-nidine. Our characterization of S. flexneri 2aMC-I and S. dysenteriae 1 MC-V revealed thattheir general properties were typical of otherminicell-producing mutants. Free MC-I andMC-V minicells were produced by aberrant celldivisions at the polar ends of these mutant bac-teria (Fig. 1). Such free minicells, readily purifiedfrom stationary-phase cultures of both mutantsby using differential rate sedimentation in linearsucrose gradients, were found to contain littleDNA (Table 1). The amounts of DNA detectedwere similar to those previously found with

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300 GEMSKI AND GRIFFIN70%

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FIG. 2. Separation ofS. flexneri M42-43MC-Imin-icells by sucrose gradient centrifugation (see the text).OD, Optical density.

TABLE 1. DNA content ofpurified minicells'DNA (mg/mg of protein)

Strain ~~~MC/WChIStrain Purified mini- Whole ratiocells cells

S. flexneri 2a MC-I 0.0022 0.45 0.0049S. dysenteriae 1 MC- 0.0014 0.47 0.0029V

aProtein concentrations were determined by using themethod of Lowry et al. (12) with crystalline bovine serumalbumin as the standard. DNA concentrations were estab-lished by employing the method of Burton (2) with herringDNA as the standard.

b MC, Purified minicells; WC, whole cells.

other minicell-producing strains (5). These lowlevels of DNA may reflect the presence of con-taminating parental cells in the suspensions andthe possibility that free MC-I and MC-V mini-cells contain plasmid DNA. Previous studies ofE. coli minicells have shown that plasmid DNAof minicell-producing strains can segregate intofree minicells (5). Since the presence of crypticplasmids in shigellae has long been known (14)and recently confirmed (11), it is possible that

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the suspensions of purified MC-I and MC-Vminicells contained such DNA.Our findings indicate that both minicell-pro-

ducing strains retain virulence properties typicalof their wild-type S. flexneri and S. dysenteriae1 parents (6). It is evident that their aberrantcell division cycle has not significantly affectedpenetration of and multiplication within epithe-lial cells of the cornea and HeLa cells. Freeminicells from S. dysenteriae MC-V were foundto contain significant levels of Shiga toxin (Table2). Preliminary studies have indicated, however,that free MC-I and MC-V minicells do not retainan ability to invade HeLa cells. The reason(s)for this remains obscure. Although studies ofother minicell systems have revealed that mini-cells possess a variety of transport, metabolic,and macromolecular synthetic systems, it is pos-sible that certain cellular functions related tothe invasive property of shigellae are lacking inminicells. The absence in minicells of chromo-somal DNA and of de novo synthesis of chro-mosomal gene-encoded RNA and protein prod-ucts may indicate that the invasive process in-volves participation of such factors. Studies ofthe penetration of virulent shigellae into Henleepithelial cells have revealed that the invadingpathogen, an active participant in the event,must possess normal physiological and syntheticfunctions (10).The recovery of virulent minicell-producing

mutants of shigellae provides an opportunity tostudy the effectiveness of purified minicells asnonreplicating oral vaccines against shigellosis.Results from a preliminary study in which rhe-sus monkeys were fed twice with 10'° purifiedMC-I minicells and then subsequently chal-lenged orally with virulent S. flexneri M4243failed to provide evidence of protection (unpub-lished observation). This pilot study did, how-ever, demonstrate the feasibility of preparinglarge quantities of minicells for further safetyand vaccination studies. Additional studies ofthe segregation of cryptic plasmids into MC-Iand MC-V minicells may provide a useful ave-nue for defining the products encoded by themand determining their role in virulence. Furtherinvestigations of the ability of purified shigellaminicells to penetrate epithelial cells and of S.dysenteriae minicells to elaborate Shiga enter-otoxin may provide some new insights into ourunderstanding of the role of these factors in thepathogenesis of shigellosis.

ACKNOWLEDGMENTSWe thank M. Judith Gemski, K. McConnell, and S. Moseley

for their technical assistance in performing these studies. Weare grateful to S. W. Rothman for helpful comments on themanuscript.

MINICELLS OF SHIGELLA SPP. 301V~~~~~~~~~~~~~~~~.4P..*1'-AW~

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-3eerio -FIG. 3. Penetration of HeLa cells by S. flexneri M42-43 MC-I (see the text).

TABLE 2. Cytotoxicity of Shiga toxin released frompurified minicells by polymyxin B

Toxin titeraIncubation Treatmenttime (mm) MC-I MC-V

2 Polymyxin B <1:5 1:680Control <1:5 <1:5

60 Polymyxin-B <1:5 1:420Control <1:5 1:20

a Toxin titer is defined as the amount of toxin thatwill cause a 50% detachment of a HeLa cell monolayerafter overnight incubation (8).

LITERATURE CITED1. Adler, H. I., W. D. Fisher, A. Cohen, and A. A. Har-

digree. 1967. Miniature Escherichia coli cells deficientin DNA. Proc. Natl. Acad. Sci. U.S.A. 57:321-326.

2. Burton, K. 1956. A study of the conditions and mecha-

nisms of the diphenylamine reaction for the calorimetricestimation of deoxyribonucleic acid. Biochem. J. 62:315-323.

3. Cerny, G., and M. Teuber. 1971. Different release ofperiplasmic versus cytoplasmic enzymes form Esche-richia coli B by polymyxin B. Arch. Mikrobiol. 78:166-179.

4. Formal, S. B., P. Gemski, Jr., L. S. Baron, and E. H.LaBrec. 1971. Chromosomal locus which controls theability of Shigella flexneri to evoke keratoconjunctivi-tis. Infect. Immun. 3:73-79.

5. Frazer, A. C., and R. Curtiss. 1975. Production, prop-erties and utility of bacterial minicells. Curr. Top. Mi-crobiol. Immunol. 69:1-84.

6. Gemski, P., Jr., and S. B. Formal. 1975. Shigellosis: aninvasive infection of the gastrointestinal tract, p. 165-169. In D. Schlessinger (ed.), Microbiology-1975.American Society of Microbiology, Washington, D.C.

7. Gemski, P., Jr., A. Takeuchi, 0. Washington, and S.B. Formal. 1972. Shigellosis due to Shigella dysenter-iae 1: relative importance of mucosal invasion versustoxin production in pathogenesis. J. Infect. Dis. 126:523-530.

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8. Gentry, M. K., and J. M. Dalrymple. 1980. Quantitativemicrotiter cytotoxicity assay for Shigella toxin. J. Clin.Microbiol. 12:361-366.

9. Giannella, R., 0. Washington, P. Gemski, and S. B.Formal. 1973. Invasion of HeLa cells by Salmonellatyphimurium: a model for study of invasiveness of sal-monella. J. Infect. Dis. 128:69-75.

10. Hale, T., and P. F. Bonventre. 1979. Shigella infectionof Henle intestinal epithelial cells: role of the bacterium.Infect. Immun. 24:879-886.

11. Kopecko, D., J. Holcombe, and S. Formal. 1979. Mo-lecular characterization of plasmids from virulent andspontaneously occurring avirulent colonial variants ofShigella flexneri. Infect. Immun. 24:580-582.

12. Lowry, 0. H., N. J. Rosebrough, A. L. Farr, and R. J.Randall. 1951. Protein measurement with the Folin

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phenol reagent. J. Biol. Chem. 193:265-275.13. O'Brien, A. D., M. R. Thompson, P. Gemski, B. P.

Doctor, and S. B. Formal. 1977. Biological propertiesof Shigella flexneri 2A toxin and its serological rela-tionship to Shigella dysenteriae 1 toxin. Infect. Immun.15:796-798.

14. Rush, M. G., C. N. Gordon, and R. C. Warner. 1969.Circular deoxyribonucleic acid from Shigella dysenter-iae Y6R. J. Bacteriol. 100:803-808.

15. Sereny, B. 1955. Experimental shigella keratoconjuncti-vitis: a preliminary report. Acta Microbiol. Acad. Sci.Hung. 2:293-296.

16. Wilkinson, R. G., P. Gemski, and B. A. D. Stocker.1972. Non-smooth mutants of Salmonella typhimu-rium: differentiation by phage sensitivity and geneticmapping. J. Gen. Microbiol. 70:527-554.